Oncolytic viruses have proved safe and effective in preclinical testing and are now in clinical trials for patients with a variety of malignant diseases. The current generation of oncolytic herpes simplex viruses (HSVs) was constructed exclusively from type 1 virus (HSV-1) and their tumor selectivity was largely achieved by targeting dividing cells. We recently constructed a novel oncolytic virus from HSV-2 that can selectively replicate in and lyse tumor cells with an activated Ras signaling pathway. Designated FusOn-H2, the mutant virus features multiple antitumor mechanisms, including the induction of cell membrane fusion (syncytia formation) and apoptosis in tumor cells, and thus has potent antitumor activity when tested in different tumor models. Nonetheless, we are well aware of the remaining barriers to successful clinical application of FusOn-H2-mediated virotherapy. For example, the antitumor effect of any oncolytic virus is greatly diminished by the host's innate immunity, which can be instantly activated during virus infection, leading us to suggest that interference with key components of the innate immune system might enhance the oncolytic effects of FusOn-H2. We also hypothesize that the unique oncolytic mechanisms of FusOn- H2 could be exploited in combination regimens with chemotherapy to further potentiate the tumor cell destruction, and that mesenchymal stem cells would function as ideal cell carriers to selectively and repeatedly deliver the oncolytic virus to tumor tissues, even in the presence of active antiviral immunity. We have proposed three specific aims to test these predictions both in vitro and in vivo, with the long-term goal of developing a potent and safe virotherapy that could be translated into a clinically useful strategy in the near future. Successful outcomes of these preclinical studies will have significant implications for the design of future clinical trials of this novel oncolytic virus, as one or more of these enhancement strategies may be implemented to potentiate this virotherapy. PUBLIC HEALTH RELEVANCE: Using viruses to kill tumor cells has shown considerable promise in the laboratory and in early clinical trials. Yet several obstacles must be overcome before this strategy gains acceptance as a safe and potent form of cancer therapy. The applicant has constructed a virus from herpes simplex virus type 2, called FusOn-H2, which has produced encouraging antitumor activity in animal tumor models. He now proposes to develop and test different strategies for improving FusOn-H2 virotherapy, with the long-term goal of translating this treatment into a clinically feasible option for patients with malignant sold tumors.